Plants perceive light signals through photoreceptors such as CRY1 to regulate growth and development. It is well-known that Arabidopsis CRY1 is a nucleocytoplasmic protein that mediates light inhibition of hypocotyl elongation in the nucleus, but the mechanisms by which CRY1 regulates root growth and functions in the cytoplasm remain poorly understood. Here, we identified eIF3G1, a subunit of the eukaryotic translation initiation factor 3 (eIF3) complex, as a CRY1-interacting protein associated with light-regulated root development. Under blue light, eif3g1 mutants showed longer primary roots, whereas eIF3G1 overexpression reduced root elongation, accompanied by corresponding changes in root apical meristem size. Differential irradiation experiments indicated that shoot illumination is required for eIF3G1-dependent root phenotypes. Transcriptome analysis revealed changes in translation-related and light-responsive genes in response to eIF3G1 perturbation. Comparison with the cry1 transcriptome revealed overlapping differentially expressed genes, including BIC1 and BIC2, and the bic1 bic2 double mutant showed reduced root elongation. Together, these findings identify eIF3G1 as a CRY1-interacting factor that contributes to the shoot-dependent regulation of root growth under blue light, suggesting that eIF3G1 may be associated with the CRY1-dependent shoot-to-root regulation of root growth.

A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two earliest field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

Plant transformation efficiency is highly dependent on species, individual genotypes, and tissue types. In maize, immature embryos are regularly used for transformation. The process relies heavily on callus development, as it is intricately associated with somatic embryogenesis and subsequent plant regeneration, both of which directly affect transformation efficiency. Immature embryos of the segregation progeny derived from the two inbred parents, a transformation-amenable line A188 and a recalcitrant line B73, can be cultured to form two primary callus types: Type I and Type II. The Type II callus grows faster and is a favorable type for regeneration. Here, Type I and II calli from the B73xA188 F2 population were genotyped by Genotyping-By-Sequencing (GBS). Quantitative trait locus (QTL) analysis of the callus type identified QTLs at chromosomes 2, 5, 6, 8, and 9. The result was largely supported by the bulk segregant RNA-seq (BSR-seq) genetic analysis using RNA from separately pooled Type I and II calli. Both analyses revealed that an allele of A188 on chromosome 6 and B73 alleles on chromosomes 2, 5, 8, and 9 promoted the formation of the Type II callus. Differentially expressed genes (DEGs) between the Type II and I F2 calli were also identified. In addition, the A188 calli developed from the same immature embryos often exhibit heterogeneous morphology, including the fast- and slow-growing callus sectors. The transcriptional comparison between the two sectors was performed to identify DEGs. Both sets of DEGs were enriched in genes involved in cell-wall organization and wax biosynthesis pathways.

The Elongator complex is conserved in a wide range of species and plays crucial roles in diverse cellular processes. We have previously shown that the Elongator protein PoElp3 was involved in the asexual development, pathogenicity, and autophagy of the rice blast fungus. In this study, we further revealed that PoElp3 functions via tRNA-mediated protein integrity. Phenotypic analyses revealed that overexpression of two of the tRNAs, tK(UUU) and tQ(UUG) could rescue the defects in ΔPoelp3 strain. TMT-based proteomic and transcriptional analyses demonstrated that 386 proteins were down-regulated in ΔPoelp3 strain compared with wild type strain Guy11, in a transcription-independent manner. Codon usage assays revealed an enrichment of Glutamine CAA-biased mRNA in the 386 proteins compared with the 70-15 genome. In addition to those reported previously, we also found that PoErp9, a sphingolipid C9-methyltransferase, was down-regulated in the ΔPoelp3strain. Through an ILV2-specific integration of PoERP9-GFP into the wild type and ΔPoelp3 strain, we were able to show that PoErp9 was positively regulated by PoElp3 translationally but not transcriptionally. Functional analyses revealed that PoErp9 was involved in the fungal growth, conidial development, pathogenicity, and TORrelated autophagy homeostasis in Pyricularia oryzae. Taken together, our results suggested that PoElp3 acts through the tRNA-mediated translational efficiency to regulate asexual development, pathogenicity, sphingolipid metabolism, and autophagy in the rice blast fungus.

Magnaporthe oryzae (syn. Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. Individual M. oryzae strains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of a Lolium strain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Contour-clamped homogeneous electric field (CHEF) karyotyping and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Optimization of a fluorescence in situ hybridization (FISH) protocol enabled single-cell karyotyping and revelation of drastic cellular variation in numbers of mini-chromosomes. In addition, rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. We found that, in contrast to Long Terminal Repeat (LTR) retrotransposons in core chromosomes, LTR retrotransposons in mini-chromosomes were more recently inserted, less methylated, and with higher G + C content. The data indicated that most LTR retrotransposons in mini-chromosomes retain high activity and have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation, which may contribute to highly dynamic mini-chromosome content in fungi.

Many pathogenic fungi display 'two-speed genome', with the fast-evolving genomic compartments enriched with repetitive sequences, particularly the transposons, which have been shown to drive the variation of pathogenicity-associated genes. Supernumerary chromosomes (SCs) are known to facilitate genomic variation in fungal pathogens, but their specific role in such processes remains understudied. In this study, we assessed the transferability of SCs between asexual Magnaporthe oryzae strains during co-culture and co-infection, and investigated their role in genome reconstruction through experimental evolution assays. We found that SCs could be horizontally transferred between M. oryzae strains and revealed frequent structural variations facilitated by SCs, including deletions, duplications, translocations, and SC-core chromosome recombinations during and after horizontal transfer. Remarkably, all observed intra- and inter-chromosome rearrangements were confined to core chromosome ends and SCs, indicating a robust role of SCs in facilitating genetic exchange within fast-evolving genomic compartments. Additionally, SC carrying the avirulence gene AvrPikE modulates M. oryzae virulence against Pikh rice through horizontal transfer, loss of whole SC, and segmental deletions. Our findings establish SCs as critical players in shaping the diversity and dynamics of the pathogenic fungal genomes, highlighting them as a cradle for the variation of pathogenicity-associated genes.

High-temperature stress, also referred to as heat stress, often has detrimental effects on plant growth and development. Phytochromes have been implicated in the regulation of plant heat-stress responses, but the role of blue-light receptors, such as cryptochromes, in plant blue-light-dependent heat-stress responses remains unclear. We found that cryptochrome 1 (CRY1) negatively regulates heat-stress tolerance (thermotolerance) in Arabidopsis. Heat stress represses CRY1 phosphorylation. Unphosphorylated CRY1 exhibits decreased activity in suppressing the interaction of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) with ELONGATED HYPOCOTYL 5 (HY5), leading to excessive degradation of HY5 under heat stress in blue light. This reduction in HY5 protein levels subsequently relieves its repression of the transcription of HY5 target genes, especially the heat-shock transcription factors. Our study thus reveals a novel mechanism by which CRY1-mediated blue-light signaling suppresses plant thermotolerance and highlights the dual function of the CRY1-COP1-HY5 module in both light-and heat-stress signaling, providing insights into how plants integrate heat stress and light signals to optimize their survival under heat stress.

The Elongator complex is conserved in a wide range of species and plays crucial roles in diverse cellular processes. We have previously shown that the Elongator protein PoELp3 was involved in the asexual development, pathogenicity, and autophagy of the rice blast fungus. In this study, we further revealed that PoElp3 functions via tRNA-mediated protein integrity. Phenotypic analyses revealed that overexpression of two of the tRNAs, tK(UUU) and tQ(UUG) could rescue the defects in ΔPoelp3 strain. TMT-based proteomic and transcriptional analyses demonstrated that 386 proteins were down-regulated in ΔPoelp3 strain compared with wild type strain Guy11, in a transcription-independent manner. Codon usage assays revealed an enrichment of Glutamine CAA-biased mRNA in the 386 proteins compared with the 70-15 genome. In addition to those reported previously, we also found that PoErp9, a sphingolipid C9-methyltransferase, was down-regulated in the ΔPoelp3 strain. Through an ILV2-specific integration of PoERP9-GFP into the wild type and ΔPoelp3 strain, we were able to show that PoErp9 was positively regulated by PoElp3 translationally but not transcriptionally. Functional analyses revealed that PoErp9 was involved in the fungal growth, conidial development, pathogenicity, and TOR-related autophagy homeostasis in P. oryzae. Taken together, our results suggested that PoElp3 acts through the tRNA-mediated translational efficiency to regulate asexual development, pathogenicity, sphingolipid metabolism, and autophagy in the rice blast fungus.

BACKGROUND: Dicer1 plays a crucial role in regulating the development and reproduction of insects. Knockout of Dicer1 causes pupal deformity, low eclosion and low fecundity in Plutella xylostella, but the mechanism behind this phenomenon is not clear. This study aims to identify differentially-expressed genes and miRNAs in the Dicer1-knockout strain (Delta PxDcr-1) and assess their impact on the reproduction and development of P. xylostella. RESULTS: The knockout of Dicer1 affected the expression of genes including the adipokinetic hormone/corazonin-related peptide receptor (PxACPR). The expression of PxACPR was upregulated, and the expression of miR-8514-5p was downregulated in Delta PxDcr-1 of P. xylostella. The dual luciferase reporter assay and pull-down assay showed that miR-8514-5p bound to PxACPR in vitro and in vivo. The expression profiles demonstrated a negative correlation between PxACPR mRNA and miR-8514-5p in different developmental stages of the wild-type strain. Both the miR-8514-5p agomir and double-stranded RNA of ACPR (dsPxACPR) injected into the pre-pupae inhibited the mRNA level of PxACPR, causing high mortality and deformity of pupae, and low fecundity and hatching rate, which were consistent with the phenotype of Delta PxDcr-1. The injection of miR-8514-5p antagomir caused a similar phenotype to the injection of miR-8514-5p agomir. Additionally, the injection of miR-8514-5p antagomir significantly rescued the phenotype caused by dsPxACPR. CONCLUSION: These results indicate that miR-8514-5p affects the development and reproduction of P. xylostella by regulating PxACPR, and the homeostasis of PxACPR expression is essential for the development and reproduction of P. xylostella. (c) 2024 Society of Chemical Industry.

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m6A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid-liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis.

BACKGROUND:Pine wood nematode (PWN; Bursaphelenchus xylophilus) is the causative agent of pine wilt disease (PWD), which is considered the most dangerous biohazard to conifer trees globally. The transmission of PWN relies on insect vectors, particularly the Japanese pine sawyer (JPS; Monochamus alternatus). However, the molecular mechanism underlying PWN-JPS assembly remains largely unknown. RESULTS:Here, we found that both geographical and gender could significantly affect the PCA (PWN carrying amount) of JPS; thus, JPS transcriptomes from diverse locations and genders were explored regard to PWN loading. Due to the shortage of genomes, we developed a full-length reference transcriptome for analyzing next-generation sequencing data. A comparative genomic study was performed, and 11 248 potential PWN-carrying associate genes (β) were nominated in JPS by using the reported genomes of PWN and non-PWN carrier insect species. Then, 151 differentially expressed transcripts (DETs), 28 of them overlapped with β, correlated with the PCA of JPS were nominated by RNA-Seq, and found that fatty acid β-oxidation might be the key factor that affected the PCA of JPS. Furthermore, JPS fatty acid β-oxidation rates were experimentally decreased using the inhibitor Etomoxir, leading to an increased PCA of JPS. Meanwhile, silencing MaCPT1 in JPS by RNA interference led to a decreased fatty acid β-oxidation rate and increased PCA of JPS. CONCLUSIONS:In conclusion, MaCPT1 was able to decrease the PWN-JPS assembly formation through the fatty acid β-oxidation of JPS. These results provide new insights for exploring the impact of PWN invasion on JPS. © 2024 Society of Chemical Industry.

Abstract Magnaporthe oryzae(syn.Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. IndividualM.oryzaestrains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of aLoliumstrain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Karyotyping analysis and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. The susceptibility to structural variation in mini-chromosomes may be associated with repetitive features and the high activity of transposable elements in mini-chromosomes, in which many intact retrotransposons were recently inserted, largely unmethylated, and likely have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation.

Magnaporthe oryzae (syn. Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. Individual M. oryzae strains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of a Lolium strain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Karyotyping analysis and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. The susceptibility to structural variation in mini-chromosomes may be associated with repetitive features and the high activity of transposable elements in mini-chromosomes, in which many intact retrotransposons were recently inserted, largely unmethylated, and likely have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation. ### Competing Interest Statement SL is the co-founder of Data2Bio, LLC. Other authors claim no competing interest.

The genomes of the fungus Magnaporthe oryzae that causes blast diseases on diverse grass species, including major crops, have indispensable core-chromosomes and may contain supernumerary chromosomes, also known as mini-chromosomes. These mini-chromosomes are speculated to provide effector gene mobility, and may transfer between strains. To understand the biology of mini-chromosomes, it is valuable to be able to detect whether a M. oryzae strain possesses a mini-chromosome. Here, we applied recurrent neural network models for classifying DNA sequences as arising from core- or mini-chromosomes. The models were trained with sequences from available core- and mini-chromosome assemblies, and then used to predict the presence of mini-chromosomes in a global collection of M. oryzae isolates using short-read DNA sequences. The model predicted that mini-chromosomes were prevalent in M. oryzae isolates. Interestingly, at least one mini-chromosome was present in all recent wheat isolates, but no mini-chromosomes were found in early isolates collected before 1991, indicating a preferential selection for strains carrying mini-chromosomes in recent years. The model was also used to identify assembled contigs derived from mini-chromosomes. In summary, our study has developed a reliable method for categorizing DNA sequences and showcases an application of recurrent neural networks in predictive genomics.

Fungi from the Pyricularia genus cause blast disease in many economically important crops and grasses, such as wheat, rice, and Cenchrus grass JUJUNCAO. Structure variation associated with the gain and loss of effectors contributes largely to the adaptive evolution of this fungus towards diverse host plants. A telomere-to-telomere genome assembly would facilitate the identification of genome-wide structural variations through comparative genomics. Here, we report a telomere-to-telomere, near-complete genome assembly of a Pyricularia penniseti isolate JC-1 infecting JUJUNCAO. The assembly consists of eight core chromosomes and two supernumerary chromosomes, named mini1 and mini2, spanning 42.1 Mb. We annotated 12,156 protein-coding genes and identified 4.54% of the genome as repetitive sequences. The two supernumerary chromosomes contained fewer genes and more repetitive sequences than the core chromosomes. Our genome and results provide valuable resources for the future study in genome evolution, structure variation and host adaptation of the Pyricularia fungus.

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing. In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching. Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved. However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity. We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9. The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences. We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in P. xylostella, which can also be used for early sex detection. By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene. Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P. xylostella, while the establishment of a heritable strain required the positioning of appropriate sites. This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

The ongoing interplay among plants, insects, and bacteria underscores the intricate balance of defense mechanisms in ecosystems. Regurgitant bacteria directly/indirectly impact plant immune responses, but the underlying mechanism is unclear. Here, we focus on the interaction between regurgitant bacteria, diamondback moth (DBM), and plant. Six culturable bacteria were isolated from DBM gut regurgitant, including three Enterobacter strains (RB1-3), Micrococcus sp. (RB4), Staphylococcus haemolyticus (RB5), and Bacillus cereus (RB6). These RB strains suppressed genes related to jasmonic acid and glucosinolate signaling pathways but had little effect on salicylic acid signaling pathway genes in Arabidopsis thaliana wounds. RB1 and RB5 inhibited DBM development on A. thaliana but not on an artificial diet. RB1 and RB5 significantly suppressed GOX genes and proteins in DBMs. However, the Pxgox2 insect mutant strain inoculated with RB1 or RB5 did not significantly affect DBM feeding on A. thaliana compared to the wild type. Six RB have been functionally identified, with RB1 and RB5 negatively regulating GOX-mediated host adaptability. The deliberate addition of RB1 and RB5 can negatively affect DBM herbivory and fitness. Our study provides a molecular basis for the further application of RB for insect pest management by modulating insect–plant interactions.

Translation initiation is a critical, rate-limiting step in protein synthesis. The eukaryotic translation initiation factor 4E (eIF4E) plays an essential role in this process. However, the mechanisms by which eIF4E-dependent translation initiation regulates plant growth and development remain not fully understood. In this study, we found that Arabidopsis eIF4E proteins are distributed in both the nucleus and cytoplasm, with only the cytoplasmic eIF4E being involved in the control of photoperiodic flowering. Genome-wide translation profiling using Ribo-tag sequencing reveals that eIF4E may regulate plant flowering by maintaining the homeostatic translation of components in the photoperiodic flowering pathway. eIF4E not only regulates the translation of flowering genes such as FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FLD) but also influences the translation of circadian genes like CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and PSEUDO-RESPONSE REGULATOR 9 (PRR9). Consistently, our results show that the eIF4E modulates the rhythmic oscillation of the circadian clock. Together, our study provides mechanistic insights into how the protein translation regulates multiple developmental processes in Arabidopsis, including the circadian clock and photoperiodic flowering.

The fungal pathogen, Magnaporthe oryzae Triticum pathotype, causing wheat blast disease was first identified in South America and recently spread across continents to South Asia and Africa. Here, we studied the genetic relationship among isolates found on the three continents.Magnaporthe oryzae strains closely related to a South American field isolate B71 were found to have caused the wheat blast outbreaks in South Asia and Africa. Genomic variation among isolates from the three continents was examined using an improved B71 reference genome and whole-genome sequences. We found strong evidence to support that the outbreaks in Bangladesh and Zambia were caused by the introductions of genetically separated isolates, although they were all close to B71 and, therefore, collectively referred to as the B71 branch. In addition, B71 branch strains carried at least one supernumerary mini-chromosome. Genome assembly of a Zambian strain revealed that its mini-chromosome was similar to the B71 mini-chromosome but with a high level of structural variation. Our findings show that while core genomes of the multiple introductions are highly similar, the mini-chromosomes have undergone marked diversification. The maintenance of the mini-chromosome and rapid genomic changes suggest the mini-chromosomes may serve important virulence or niche adaptation roles under diverse environmental conditions.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.